There is growing
interest in
digital PCR (dPCR) because technological progress makes it a practical
and increasingly affordable technology. dPCR allows the precise
quantification of nucleic acids, facilitating the measurement of small
percentage differences and quantification of rare variants. dPCR may
also be more reproducible and less susceptible to inhibition than
quantitative real-time PCR (qPCR). Consequently, dPCR has the potential
to have a substantial impact on research as well as diagnostic
applications. However, as with qPCR, the ability to perform robust
meaningful experiments requires careful design and adequate controls.
To assist independent evaluation of experimental data, comprehensive
disclosure of all relevant experimental details is required. To
facilitate this process we present the Minimum Information for
Publication of Quantitative Digital PCR Experiments guidelines. This
report addresses known requirements for dPCR that have already been
identified during this early stage of its development and commercial
implementation. Adoption of these guidelines by the scientific
community will help to standardize experimental protocols, maximize
efficient utilization of resources, and enhance the impact of this
promising new technology.

Introduction

Definition:

Digital PCR (dPCR) is a
refinement of conventional PCR methods that can be used to directly
quantify and clonally amplify nucleic acids (including DNA, cDNA,
methylated DNA, or RNA). The key difference between dPCR and
traditional PCR lies in the method of measuring nucleic acids amounts,
with the former being a more precise method than PCR. PCR carries out
one reaction per single sample. dPCR also carries out a single reaction
within a sample, however the sample is separated into a large number of
partitions and the reaction is carried out in each partition
individually. This separation allows a more reliable collection and
sensitive measurement of nucleic acid amounts. The method has been
demonstrated as useful for studying variations in gene sequences - such
as copy number variants, point mutations, and
it is routinely used for clonal amplification of samples for
"next-generation sequencing."

PCR
Basics:

The PCR method is used
to quantify nucleic acids by amplifying a nucleic acid molecule with
the enzyme DNA polymerase. Conventional PCR is based on the theory that
amplification is exponential. Therefore, nucleic acids may be
quantified by comparing the number of amplification cycles and amount
of PCR end-product to those of a reference sample. However, many
factors complicate this calculation, creating uncertainties and
inaccuracies.

These factors include the following:

Initial amplification cycles may not be exponential

PCR amplification eventually plateaus after an
uncertain number of cycles

Digital PCR overcomes
the difficulties of conventional PCR. With
dPCR, a sample is partitionedso that individual nucleic acid molecules
within the sample are localized and concentrated within many separate
regions. The partitioning of the sample allows one to count the
molecules by estimating according to Poisson. As a result, each part
will contain "0" or "1" molecules, or a negative or positive reaction,
respectively. After PCR amplification, nucleic acids may be quantified
by counting the regions that contain PCR end-product, positive
reactions.

In conventional PCR,
starting copy number is proportional to the number of PCR amplification
cycles. dPCR, however, is not dependent on the number of amplification
cycles to determine the initial sample amount, eliminating the reliance
on uncertain exponential data to quantify target nucleic acids and
providing absolute quantification.

Development:

The dPCR concept was
conceived in 1992 by Sykes et al. using nested PCR. An important
development occurred in 1995 with co-inventions by Brown at Cytonix and
Silver at the National Institutes of Health of single-step
quantitization and sequencing methods employing nano-scale arrays and
localized clonal colonies using capillaries, gels, affinity
surfaces/particles and immiscible fluid containments, resulting in a
1997 U. S. Patent (U. S. Patent 6,143,496)and subsequent
divisional and continuation patents.
Vogelstein and Kinzler further developed the concept by quantifying
KRAS mutations in stool DNA from colorectal cancer patients. Digital
PCR has been shown to be a promising surveillance tool for illnesses
such as cancer. Significant additional developments have included using
emulsion beads for digital PCR by Dressman and colleagues. Digital PCR
has many other applications, including detection and quantitization of
low-level pathogens, rare genetic sequences, gene expression in single
cells, and the clonal amplification of nucleic acids (cPCR or clonal
PCR) for the identification and sequencing of mixed nucleic acids
samples or fragments. It has also proved useful for the analysis of
heterogeneous methylation.

Digital PCR has many
potential applications, including the detection and quantification of
low-level pathogens, rare genetic sequences, copy number variations,
and relative gene expression in single cells. Clonal amplification
enabled by single-step digital PCR is a key factor in reducing the time
and cost of many of the "next-generation sequencing" methods and hence
enabling personal genomics.

Application of digital PCR
for Absolute Quantitation
Digital PCR is quantitative PCR method that can be used to measure
absolute quantitation. In this technique, the number of positive and
negative amplification reactions is used to the determine precise
measurement of target concentration.

dPCR
Applications:

Absolute Quantification of Viral Load

Absolute Quantification of Nucleic Acid Standards

Absolute Quantification of Next-Gen Sequencing
Libraries

Rare Allele Detection

Low-Fold Copy Number Discrimination

Enrichment and Separation of Mixtures

Summary
Advantages of Digital PCR:

No need to rely on references or standards

Desired precision can be achieved by increasing
total number of PCR replicates

Highly tolerant to inhibitors

Capable of analyzing complex mixtures

Unlike traditional qPCR, digital PCR provides a
linear
response to the number of copies present to allow for small fold change
differences to be detected

When calculating the "absolute" results of your real-time PCR (qPCR)
experiment,
you can use either digital PCR method
or
classical standard curve based
"absolute quantification". More
info at Life Technologies

Absolute
Quantification at a Glance

Absolute Quantification
(Digital PCR Method)

Absolute Quantification
(Standard Curve Method)

Overview

In
absolute quantification using Digital PCR, no known standards are
needed. The target of interest can be directly quantified with
precision determined by number of digital PCR replicates.

In
absolute quantification using the Standard Curve Method, you quantitate
unknowns based on a known quantity. First you create a standard curve;
then you compare unknowns to the standard curve and extrapolate a value.

Example

Quantify
copies of rare allele present in heterogenous mixtures.

Count the number of cell equivalents in sample by targeting genomic DNA.

Determine absolute number of viral copies present in a given sample
without reference to a standard.

Correlating
viral copy number with a disease state.

Absolute
Quantification Using the Digital PCR Method

Digital
PCR works by partitioning a sample into many individual real-time PCR
reactions; some portion of these reactions contain the target molecule
(positive) while others do not (negative). Following PCR analysis, the
fraction of negative answers is used to generate an absolute answer for
the exact number of target molecules in the sample, without reference
to standards or endogenous controls.

Figure 1:
Digital PCR uses the ratio of positive (White) to negative (Black) PCR
reactions
to count the number of target molecules.

Absolute
Quantification Using the Standard Curve
Method

The
standard curve method for absolute quantification is similar to the
standard curve method for relative quantification, except the absolute
quantities of the standards must first be known by some independent
means.

Critical
Guidelines
The guidelines below are critical for proper use of the standard curve
method for absolute quantification:

It
is important that the DNA or RNA be a single, pure species. For
example, plasmid DNA prepared from E. coli often is contaminated with
RNA, which increases the A260 measurement and inflates the copy number
determined for the plasmid.

Accurate pipetting is required
because the standards must be diluted over several orders of magnitude.
Plasmid DNA or in vitro transcribed RNA must be concentrated in order
to measure an accurate A260 value. This concentrated DNA or RNA must
then be diluted 106–1012 -fold to be at a concentration similar to the
target in biological samples.

The stability of the diluted
standards must be considered, especially for RNA. Divide diluted
standards into small aliquots, store at –80 °C, and thaw only once
before use.

It is generally not possible to use DNA as a
standard for absolute quantification of RNA because there is no control
for the efficiency of the reverse transcription step.

Standards
The absolute quantities of the standards must first be known by some
independent means. Plasmid DNA and in vitro transcribed RNA are
commonly used to prepare absolute standards. Concentration is measured
by A260 and converted to the number of copies using the molecular
weight of the DNA or RNA.

Quantitative PCR (qPCR)
has
become the gold standard technique to measure cDNA and gDNA levels but
the resulting data can be highly variable, artifactual and
non-reproducible without appropriate verification and validation of
both samples and primers. The root cause of poor quality data is
typically associated with inadequate dilution of residual protein and
chemical contaminants that variably inhibit Taq polymerase and primer
annealing. The most susceptible, frustrating and often most interesting
samples are those containing low abundant targets with small expression
differences of 2-fold or lower. Here, Droplet Digital PCR (ddPCR) and
qPCR platforms were directly compared for gene expression analysis
using low amounts of purified, synthetic DNA in well characterized
samples under identical reaction conditions. We conclude that for
sample/target combinations with low levels of nucleic acids (Cq ≥ 29)
and/or variable amounts of chemical and protein contaminants, ddPCR
technology will produce more precise, reproducible and statistically
significant results required for publication quality data. A stepwise
methodology is also described to choose between these complimentary
technologies to obtain the best results for any experiment

Digital
PCR Using
the OpenArray® Real-Time PCR SystemMore
info at Life Technologies
Digital PCR is a new approach to nucleic acid detection and
quantification, which is a different method of absolute quantification
and rare allele detection relative to conventional qPCR.

Digital PCR works by partitioning a sample into many individual
real-time PCR reactions; some portion of these reactions contain the
target molecule (positive) while others do not (negative). Following
PCR analysis, the fraction negative answers is used to generate an
absolute answer for the exact number of target molecules in the sample,
without reference to standards or endogenous controls.

The OpenArray® Real-Time PCR System enables digital PCR experiments
at a scale previously unattainable—in a single day, one user can
generate >36,000 digital PCR data points on the OpenArray®
Real-Time PCR System, without the use of robotics. Other features of
the system include:

Flexible—enables use of your existing assays and has
the capacity to test from one to 48 assay/sample dilutions per plate

Wide dynamic range—with as few as 64 data points per
replicate group, a dilution series can easily be loaded into the
TaqMan® OpenArray® Digital PCR Plate, expanding the range of
sample concentrations which can be analyzed to produce a digital answer.

Combining two digital PCR technology, Crystal
Digital™ PCR is Stilla
Technologies next generation solution that delivers results in 2h30
with less than 5 minutes hands-on time. The heart of Stilla’s Naica
System™ is the microfluidic Sapphire Chip, fully integrating the 3
steps of Digital PCR (droplet formation, amplification and readout) in
a single consumable. Using the Sapphire Chips, the sample is
partitioned into a droplet crystal, i.e. a 2D monolayer of 30 000
droplets. The partitioning and amplification are both performed by the
Naica Geode™ thermocycler while the Naica Prism3™ instrument offers
high multiplexing capability by visualizing targets through 3
fluorescent channels (blue, red and green). Once the image acquisition
has been performed, Stilla’s Crystal Miner™ analysis software enables
the automatic identification of positive and negative droplets with an
intuitive visual inspection and analysis of the Digital PCR experiment.
From the number of droplets in each population, it gives the absolute
concentration of the target sequences.

RainDance
Technologies RainDrop Digital PCR System shifts the PCR
paradigm from a single color per marker approach to a more scalable and
precise multi-color and intensity-per-marker method. This novel
approach increases sensitivity by generating between 1 million and 10
million pico-liter sized droplets per lane, which is a 500 – 10,000x
improvement over existing PCR methods. Since each droplet encapsulates
a single molecule, researchers can quickly determine the absolute
number of droplets containing specific target DNA and compare that to
the amount of droplets with normal, background wild-type DNA. The
RainDrop System is the third generation of RainDance’s droplet-based
PCR instrumentation and uses the fourth generation of the company’s
microfluidic chips.

The
RainDrop System supports a number of important applications
including low-frequency tumor allele detection, gene expression, copy
number variation, and SNP measurement.

Advantages:

Superior sensitivity: Detect 1 mutant amongst
250,000 wild-type molecules with a lower limit of detection of 1 in
more than 1,000,000.

Unprecedented multiplexing: Conduct up to 10
tests
or more on the same sample using the single molecule multi-color
detection technique.

Greater study design flexibility: Optimize
number of
PCR reactions based on your sensitivity AND multiplex requirements.

New digital PCR reporting
guidelines for molecular diagnostics.National Measurement System
LGC scientists have collaborated on the development of best practice
guidelines for the reporting of digital PCR data. Digital PCR is an
emerging tool for DNA analysis showing great promise in new challenging
areas of clinical diagnostics. These reporting guidelines provide a
gold standard checklist of experimental information that should be
included in all digital PCR publications to enable the research
community to review and compare.

Evaluation
of digital PCR for clinical diagnostics

Polymerase
chain reaction (PCR), the method that amplifies DNA sequences by
multiple rounds of thermal cycling, has been used extensively
throughout molecular research over the last 25 years. It has many
different applications including gene expression analysis, DNA mutation
detection, cloning and sequencing and, as a result, has been widely
used in clinical medicine, forensics and biological research. In 1999,
cancer research pioneers Kenneth Kinzler and Bert Vogelstein of Johns
Hopkins University (Baltimore), modified the standard PCR method in
order to improve its sensitivity for cancer diagnostics. They named
this method digital PCR.

The main principle of digital PCR is
that a single sample is split into many fractions, all of which are
subsequently analysed by a standard PCR method (Figure 1). The sample
is fractionated by the simple process of dilution so that each fraction
contains approximately one copy of DNA template or less. By isolating
individual DNA templates this process effectively enriches DNA
molecules that were present at very low levels in the original sample.
Therefore, this method has applications for the detection of DNA
mutations (or any other DNA/RNA targets) that are present at very low
levels relative to a high background of normal DNA, for example the
early stages of cancer development. The other major feature of digital
PCR is that data are recorded as positive or negative (i.e. generation
of an amplification product or not). Hence, the individual readout
signals are qualitative or ‘digital’ in nature.

Figure 1 – The
principle of digital PCR.
a) Conventional PCR: The sample contains a heterogeneous mixture of DNA
molecules where the target molecule (e.g. DNA mutation) is present at
very low levels relative to a large background of normal DNA.
Therefore, an average signal is acquired. b) Digital PCR: The sample is
split into many fractions by dilution (approximately 1 copy of DNA or
less per fraction) prior to PCR, thereby, enriching minority targets
within individual reactions

Digital
PCR can be performed
manually, but it is labour-intensive, prone to pipetting errors and
replication levels are limited by the format of plate used (i.e. 96 or
384 well). Alternative approaches to the multi-well plate method are
now emerging. One of these is the use of a microfluidic sample handling
system to split one sample into hundreds of individual reactions
chambers which reside on an ‘integrated fluidic circuit’ or chip
(Figure 2). This method also involves miniaturisation of the PCR
whereby reactions are performed in nl volumes on the chip compared with
ml volumes typically used in conventional PCR. This miniaturised
process allows performance of close to 10000 individual PCR reactions
per chip.

The
purpose of this Chemical and Biological Metrology Programme project
(DX3 - Performance standards for early diagnostic test methods)
is to evaluate the application of sensitive emerging nucleic acid
detection methods, such as digital PCR, for clinical diagnostics by
investigating the reproducibility and robustness of measurements with
regard to detection and quantitation of low copy number/minority
biomarkers compared with the current gold standard method (i.e.
conventional real time PCR).

Leukemia
represents a relevant
case study as it provides a difficult diagnostic challenge that
requires detection of low numbers of leukemic cells amongst a majority
of normal blood cells. Proof-of-principle experiments at LGC have
measured the expression levels of the Bcr-Abl fusion transcript in a
leukemia cell line. Figure 3 illustrates data from the BioMark digital
chip. Panels A to E show decreasing expression levels (i.e.
fewer positive samples) from a 2-fold serially diluted sample. Mixed
leukemia/normal samples at different ratios are now being analysed to
mimic the in vivo situation prior to the analysis of real
clinical samples.

Figure 3 – An
example of mRNA expression data generated by the Fluidigm
BioMark digital PCR. mRNA
expression of Bcr-Abl was determined using a Taqman ™ real time PCR
assay and cDNA from the K562 leukemia cell line. Panel A-E represents a
2-fold dilution series of a cDNA sample. Panel F represents a negative
control. Each panel consists of 765 individual PCR reactions which were
analytical replicates of the same original sample. Red dots represent
individual reactions where PCR products were detected. Red dots are
simply counted per panel to give the total copy number of template in
the sample (note: a correction factor is applied to account for
occasional wells that may contain >1 copy/well). Numbers down the
right-hand side represent estimated RNA copy number previously
determined by conventional real time PCR using Bcr-Abl standards as
well as the experimental copy number determined by digital PCR.

The
advent of new commercial systems that facilitate the ease of use of
digital PCR will further promote the application of this powerful
method in other clinical and experimental research applications such as
the detection of cancer biomarkers in body fluids such as blood, urine
and sputum, as well as pre-natal screening biomarkers in maternal
blood. Such methods hold promise towards non-invasive diagnostics.

Recent reports have indicated that digital PCR may be useful for the
noninvasive detection of fetal aneuploidies by the analysis of
cell-free DNA and RNA in maternal plasma or serum. In this review we
provide an insight into the underlying technology and its previous
application in the determination of the allelic frequencies of
oncogenic alterations in cancer specimens. We also provide an
indication of how this new technology may prove useful for the
detection of fetal aneuploidies and single gene Mendelian disorders.Non-invasive
prenatal diagnosis by single molecule counting technologies
Chiu RW, Cantor CR, Lo YM.
Trends Genet. 2009 Jul;25(7): 324-331
Centre for Research into Circulating Fetal Nucleic Acids, Li Ka Shing
Institute of Health Sciences, Department of Chemical Pathology, The
Chinese University of Hong Kong, Prince of Wales Hospital, 30-32 Ngan
Shing Street, Shatin, New Territories, Hong Kong SAR, China.

Non-invasive prenatal
diagnosis of fetal chromosomal aneuploidies and monogenic diseases by
analysing fetal DNA present in maternal plasma poses a challenging
goal. In particular, the presence of background maternal DNA interferes
with the analysis of fetal DNA. Using single molecule counting methods,
including digital PCR and massively parallel sequencing, many of the
former problems have been solved. Digital mutation dosage assessment
can detect the number of mutant alleles a fetus has inherited from its
parents for fetal monogenic disease diagnosis, and massively parallel
plasma DNA sequencing enables the direct detection of fetal chromosomal
aneuploidies from maternal plasma. The analytical power of these
methods, namely sensitivity, specificity, accuracy and precision,
should catalyse the eventual clinical use of non-invasive prenatal
diagnosis.Digital
polymerase chain reaction;
new diagnostic opportunitiesEuropean
Pharmaceutical Review - Genomics page 7-9; published
22 February 2010Jim Huggett 1 & Daniel J
Scott 21 Molecular and Cell Biology, LGC; 2 Project
Manager, Research and Technology Division, LGC

LGC is an international science-based company located in South West
London. A progressive and innovative enterprise, LGC operatesin
socially responsible fields underpinning the health, safety and
security of the public, and regulation and enforcement for UKgovernment
departments and blue chip clients. Our products and services enable our
customers to have a sound basis on whichto base their scientific and
commercial decisions or conformity to international statutory and
regulatory standards. DNA diagnostics gets
digitized
by Mikael Kubista and Anders StahlbergDrug
Discovery Wold - Fall 2011

Quantitative real-time PCR (qPCR) has during the last two decades
emerged as the preferred technology for nucleic acid analysis in
routine as well as in research. qPCR has the sensitivity to detect a
single molecule, the speciﬁcity to differentiate targets by a single
nucleotide, and, because of its exponential nature, virtually unlimited
dynamic range.

Since its introduction on the commercial market little more than 10
years ago,real-time PCR has become the main technical platform for
nucleic aciddetection in research and development, as well as in
routine diagnostics. In2007 the real-time PCR market revenue in the US
was estimated at $740million with annual growth of more than 10%. In
this article latestdevelopments and future expectations are presented.Video on Digital PCR
by TATAA BiocenterThe video on Digital PCR describing latest platform at
TATAA Biocenter. http://www.youtube.com/watch?v=qdFOpRbrYE0&noredirect=1
For more information how to do dPCR at TATAA Biocenter see http://www.tataa.com/Services/Projects.html#Digital

Limiting dilution
analysis (LDA)1has gained widespread accept-ance as a tool for
quantifyingcells that possess observablefunctional activities.
Thoroughly plannedtitration experiments can produce straight-forward
and interpretable single-hit kinetics,whereas analyses of
unfractionated cellpopulations over a broader dilution rangeresult in
data that deviate from linearity anddo not adhere to all-or-none
functionality(e.g. virgin and memory CD4T cells2andothers3–10).
However, by studying the factorsthat cause the deviation from
linearity, theinteractions between different cell types inthe
population can be identified and charac-terized. As a corollary, it
follows that, alongwith quantification of desired cells, LDA al-lows an
analysis of the regulatory processesthat underlie an observed activity.

An approach for
determination of hepatitis C virus (HCV) quasispecies by end-point
limiting-dilution real-time PCR (EPLD-PCR) is described. It involves
isolation of individual coexisting sequence variants of the
hypervariable region 1 (HVR1) of the HCV genome from serum specimens
using a limiting-dilution protocol. EPLD-PCR applied to an HCV outbreak
study provided insights into the epidemiological relationships between
incident and chronic cases. When applied to samples from a longitudinal
study of infected patients, HVR1 sequences from each sampling
time-point were observed to group as distinct phylogenetic clusters.
Melting peak analysis conducted on EPLD-PCR products generated from
these patients could be used for evaluation of HVR1 sequence
heterogeneity without recourse to clonal sequencing. Further, to better
understand the mechanism of single-molecule PCR, experiments were
conducted under optimal and suboptimal annealing temperatures. Under
all temperature conditions tested, HVR1 variants from the major
phylogenetic clusters of quasispecies could be amplified, revealing
that successful HVR1 quasispecies analysis is not contingent to
dilution of starting cDNA preparations to a single-molecule state. It
was found that EPLD-PCR conducted at suboptimal annealing temperatures
generated distributions of unique-sequence variants slightly different
from the distribution obtained by PCR conducted at the optimal
temperature. Hence, EPLD-PCR conditions can be manipulated to access
different subpopulations of HCV HVR1 quasispecies, thus, improving the
range of the quasispecies detection. Although EPLD-PCR conducted at
different conditions detect slightly different quasispecies
populations, as was shown in this study, the resulted samples of
quasispecies are completely suitable for molecular epidemiological
investigation in different clinical and epidemiological settings.

The identification of
predefined mutations expected to be present in a minor fraction of a
cell population is important for a variety of basic research and
clinical applications. Here, we describe an approach for transforming
the exponential, analog nature of the PCR into a linear, digital signal
suitable for this purpose. Single molecules are isolated by dilution
and individually amplified by PCR; each product is then analyzed
separately for the presence of mutations by using fluorescent probes.
The feasibility of the approach is demonstrated through the detection
of a mutant ras oncogene in the stool of patients with colorectal
cancer. The process provides a reliable and quantitative measure of the
proportion of variant sequences within a DNA sample.

We monitored PCR in
volumes of the order of 10 nl in glass microcapillaries using a
fluorescence energy transfer assay in which fluorescence increases if
product is made due to template-dependent nucleolytic degradation of an
internally quenched probe (TaqMan assay). This assay detected single
starting template molecules in dilutions of genomic DNA. The results
suggest that it may be feasible to determine the number of template
molecules in a sample by counting the number of positive PCRs in a set
of replicate reactions using terminally diluted sample. Since the assay
system is closed and potentially automatable, it has promise for
clinical applications.Digital polymerase chain reaction for
characterisation of DNA reference materials
Somanath Bhat, , Kerry R. Emslie
Biomolecular Detection and Quantification; Available online 3 May 2016

Accurate,
reliable and reproducible quantification of nucleic acids (DNA/RNA) is
important for many diagnostic applications and in routine laboratory
testing, for example, for pathogen detection and detection of
genetically modified organisms in food. To ensure reliable nucleic acid
measurement, reference materials (RM) that are accurately characterised
for quantity of target nucleic acid sequences (in copy number or copy
number concentration) with a known measurement uncertainty are needed.
Recently developed digital polymerase chain reaction (dPCR) technology
allows absolute and accurate quantification of nucleic acid target
sequences without need for a reference standard. Due to these
properties, this technique has the potential to not only improve
routine quantitative nucleic acid analysis, but also to be used as a
reference method for certification of nucleic acid RM. The article
focuses on the use and application of both dPCR and RMs for accurate
quantification.

Digital PCR represents an example of the power of PCR and provides
unprecedented opportunities for molecular genetic analysis in cancer.
The technique is to amplify a single DNA template from minimally
diluted samples, therefore generating amplicons that are exclusively
derived from one template and can be detected with different
fluorophores or sequencing to discriminate different alleles (e.g.,
wild type vs. mutant or paternal vs. maternal alleles). Thus, digital
PCR transforms the exponential, analog signals obtained from
conventional PCR to linear, digital signals, allowing statistical
analysis of the PCR product. Digital PCR has been applied in
quantification of mutant alleles and detection of allelic imbalance in
clinical specimens, providing a promising molecular diagnostic tool for
cancer detection. The scope of this article is to review the principles
of digital PCR and its practical applications in cancer research and in
the molecular diagnosis of cancer.Microfluidics
digital PCR reveals a higher than expected fraction of fetal DNA in
maternal plasma
Lun FM, Chiu RW, Allen Chan KC, Yeung Leung T, Kin Lau T, Dennis Lo YM.
Clin Chem. 2008 Oct;54(10): 1664-1672.
Centre for Research into Circulating Fetal Nucleic Acids, Li Ka Shing
Institute of Health Sciences, The Chinese University of Hong Kong,
Shatin, New Territories, Hong Kong.

BACKGROUND: The precise measurement
of cell-free fetal DNA in maternal
plasma facilitates noninvasive prenatal diagnosis of fetal chromosomal
aneuploidies and other applications. We tested the hypothesis that
microfluidics digital PCR, in which individual fetal-DNA molecules are
counted, could enhance the precision of measuring circulating fetal DNA.

METHODS: We first determined whether
microfluidics digital PCR,
real-time PCR, and mass spectrometry produced different estimates of
male-DNA concentrations in artificial mixtures of male and female DNA.
We then focused on comparing the imprecision of microfluidics digital
PCR with that of a well-established nondigital PCR assay for measuring
male fetal DNA in maternal plasma.

RESULTS: Of the tested platforms,
microfluidics digital PCR
demonstrated the least quantitative bias for measuring the fractional
concentration of male DNA. This assay had a lower imprecision and
higher clinical sensitivity compared with nondigital real-time PCR.
With the ZFY/ZFX assay on the microfluidics digital PCR platform, the
median fractional concentration of fetal DNA in maternal plasma was
> or =2 times higher for all 3 trimesters of pregnancy than
previously reported.

CONCLUSIONS: Microfluidics digital
PCR represents an improvement over
previous methods for quantifying fetal DNA in maternal plasma, enabling
diagnostic and research applications requiring precise quantification.
This approach may also impact other diagnostic applications of plasma
nucleic acids, e.g., in oncology and transplantation.Digital
PCR for the molecular detection of fetal chromosomal aneuploidy

Trisomy 21 is the most
common reason that women opt for prenatal
diagnosis. Conventional prenatal diagnostic methods involve the
sampling of fetal materials by invasive procedures such as
amniocentesis. Screening by ultrasonography and biochemical markers
have been used to risk-stratify pregnant women before definitive
invasive diagnostic procedures. However, these screening methods
generally target epiphenomena, such as nuchal translucency, associated
with trisomy 21. It would be ideal if noninvasive genetic methods were
available for the direct detection of the core pathology of trisomy 21.
Here we outline an approach using digital PCR for the noninvasive
detection of fetal trisomy 21 by analysis of fetal nucleic acids in
maternal plasma. First, we demonstrate the use of digital PCR to
determine the allelic imbalance of a SNP on PLAC4 mRNA, a
placenta-expressed transcript on chromosome 21, in the maternal plasma
of women bearing trisomy 21 fetuses. We named this the digital RNA SNP
strategy. Second, we developed a nonpolymorphism-based method for the
noninvasive prenatal detection of trisomy 21. We named this the digital
relative chromosome dosage (RCD) method. Digital RCD involves the
direct assessment of whether the total copy number of chromosome 21 in
a sample containing fetal DNA is overrepresented with respect to a
reference chromosome. Even without elaborate instrumentation, digital
RCD allows the detection of trisomy 21 in samples containing 25% fetal
DNA. We applied the sequential probability ratio test to interpret the
digital PCR data. Computer simulation and empirical validation
confirmed the high accuracy of the disease classification algorithm.Noninvasive
prenatal diagnosis of fetal chromosomal aneuploidies by
maternal plasma nucleic acid analysis

BACKGROUND: The discovery of
circulating cell-free fetal nucleic acids
in maternal plasma has opened up new possibilities for noninvasive
prenatal diagnosis. The potential application of this technology for
the noninvasive prenatal detection of fetal chromosomal aneuploidies is
an aspect of this field that is being actively investigated. The main
challenge of work in this area is the fact that cell-free fetal nucleic
acids represent only a minor fraction of the total nucleic acids in
maternal plasma. Methods and

RESULTS: We performed a review of
the literature, which revealed that
investigators have applied methods based on the physical and molecular
enrichment of fetal nucleic acid targets from maternal plasma. The
former includes the use of size fractionation of plasma DNA and the use
of the controversial formaldehyde treatment method. The latter has been
achieved through the development of fetal epigenetic and fetal RNA
markers. The aneuploidy status of the fetus has been explored through
the use of allelic ratio analysis of plasma fetal epigenetic and RNA
markers. Digital PCR has been shown to offer high precision for allelic
ratio and relative chromosome dosage analyses.

CONCLUSIONS: After a decade of work,
the theoretical and practical
feasibility of prenatal fetal chromosomal aneuploidy detection by
plasma nucleic acid analysis has been demonstrated in studies using
small sample sets. Larger scale independent studies will be needed to
validate these initial observations. If these larger scale studies
prove successful, it is expected that with further development of new
fetal DNA/RNA markers and new analytical methods, molecular noninvasive
prenatal diagnosis of the major chromosomal aneuploidies could become a
routine practice in the near future.

STUDY DESIGN: Twenty-four
amniocentesis and 16 chorionic villus samples were used for
microfluidic digital PCR analysis. Three thousand and sixty PCR
reactions were performed for each of the target chromosomes (X, Y, 13,
18, and 21), and the number of single molecule amplifications was
compared to a reference. The difference between target and reference
chromosome counts was used to determine the ploidy of each of the
target chromosomes.

RESULTS: Digital PCR accurately
identified all cases of fetal trisomy (3 cases of trisomy 21, 3 cases
of trisomy 18, and 2 cases of triosmy 13) in the 40 specimens analyzed.
The remaining specimens were determined to have normal ploidy for the
chromosomes tested.

BACKGROUND: Next-generation DNA
sequencing on the 454, Solexa, and SOLiD platforms requires absolute
calibration of the number of molecules to be sequenced. This
requirement has two unfavorable consequences. First, large amounts of
sample-typically micrograms-are needed for library preparation, thereby
limiting the scope of samples which can be sequenced. For many
applications, including metagenomics and the sequencing of ancient,
forensic, and clinical samples, the quantity of input DNA can be
critically limiting. Second, each library requires a titration
sequencing run, thereby increasing the cost and lowering the throughput
of sequencing.

RESULTS: We demonstrate the use of
digital PCR to accurately quantify 454 and Solexa sequencing libraries,
enabling the preparation of sequencing libraries from nanogram
quantities of input material while eliminating costly and
time-consuming titration runs of the sequencer. We successfully
sequenced low-nanogram scale bacterial and mammalian DNA samples on the
454 FLX and Solexa DNA sequencing platforms. This study is the first to
definitively demonstrate the successful sequencing of picogram
quantities of input DNA on the 454 platform, reducing the sample
requirement more than 1000-fold without pre-amplification and the
associated bias and reduction in library depth.

CONCLUSION: The digital PCR assay
allows absolute quantification of sequencing libraries, eliminates
uncertainties associated with the construction and application of
standard curves to PCR-based quantification, and with a coefficient of
variation close to 10%, is sufficiently precise to enable direct
sequencing without titration runs.

METHODS: We used a nanofluidic
digital PCR array platform and 16 cell lines and 20 samples of genomic
DNA from resected tumors (stages I-III) to quantify the relative
numbers of copies of the EGFR gene and to detect mutated EGFR alleles
in lung cancer. We assessed the relative number of EGFR gene copies by
calculating the ratio of the number of EGFR molecules (measured with a
6-carboxyfluorescein-labeled Scorpion assay) to the number of molecules
of the single-copy gene RPP30 (ribonuclease P/MRP 30kDa subunit)
(measured with a 6-carboxy-X-rhodamine-labeled TaqMan assay) in each
panel. To assay for the EGFR L858R (exon 21) mutation and exon 19
in-frame deletions, we used the ARMS and Scorpion technologies in a
DxS/Qiagen EGFR29 Mutation Test Kit for the digital PCR array.

Most cancer genomes are characterized by the gain or loss of copies of
some sequences through deletion, amplification or unbalanced
translocations. Delineating and quantifying these changes is important
in understanding the initiation and progression of cancer, in
identifying novel therapeutic targets, and in the diagnosis and
prognosis of individual patients. Conventional methods for measuring
copy-number are limited in their ability to analyse large numbers of
loci, in their dynamic range and accuracy, or in their ability to
analyse small or degraded samples. This latter limitation makes it
difficult to access the wealth of fixed, archived material present in
clinical collections, and also impairs our ability to analyse small
numbers of selected cells from biopsies. Molecular copy-number counting
(MCC), a digital PCR technique, has been used to delineate a
non-reciprocal translocation using good quality DNA from a renal
carcinoma cell line. We now demonstrate microMCC, an adaptation of MCC
which allows the precise assessment of copy number variation over a
significant dynamic range, in template DNA extracted from
formalin-fixed paraffin-embedded clinical biopsies. Further, microMCC
can accurately measure copy number variation at multiple loci, even
when applied to picogram quantities of grossly degraded DNA extracted
after laser capture microdissection of fixed specimens. Finally, we
demonstrate the power of microMCC to precisely interrogate cancer
genomes, in a way not currently feasible with other methodologies, by
defining the position of a junction between an amplified and
non-amplified genomic segment in a bronchial carcinoma. This has
tremendous potential for the exploitation of archived resources for
high-resolution targeted cancer genomics and in the future for
interrogating multiple loci in cancer diagnostics or prognostics.Single-molecule genomics
McCaughan F, Dear PH.
J Pathol. 2010 Jan;220(2):297-306.
MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK.

The term 'single-molecule genomics' (SMG) describes a group of
molecular methods in which single molecules are detected or sequenced.
The focus on the analysis of individual molecules distinguishes these
techniques from more traditional methods, in which template DNA is
cloned or PCR-amplified prior to analysis. Although technically
challenging, the analysis of single molecules has the potential to play
a major role in the delivery of truly personalized medicine. The two
main subgroups of SMG methods are single-molecule digital PCR and
single-molecule sequencing. Single-molecule PCR has a number of
advantages over competing technologies, including improved detection of
rare genetic variants and more precise analysis of copy-number
variation, and is more easily adapted to the often small amount of
material that is available in clinical samples. Single-molecule
sequencing refers to a number of different methods that are mainly
still in development but have the potential to make a huge impact on
personalized medicine in the future.Microfluidic
digital PCR enables multigene analysis of individual environmental
bacteria
Ottesen EA, Hong JW, Quake SR, Leadbetter JR.
Science. 2006 314(5804): 1464-1467
Division of Biology, California Institute of Technology, Pasadena, CA
91125, USA.

Gene inventory and metagenomic techniques have allowed rapid
exploration of bacterial diversity and the potential physiologies
present within microbial communities. However, it remains nontrivial to
discover the identities of environmental bacteria carrying two or more
genes of interest. We have used microfluidic digital polymerase chain
reaction (PCR) to amplify and analyze multiple, different genes
obtained from single bacterial cells harvested from nature. A gene
encoding a key enzyme involved in the mutualistic symbiosis occurring
between termites and their gut microbiota was used as an experimental
hook to discover the previously unknown ribosomal RNA-based species
identity of several symbionts. The ability to systematically identify
bacteria carrying a particular gene and to link any two or more genes
of interest to single species residing in complex ecosystems opens up
new opportunities for research on the environment.Concordance among digital gene expression,
microarrays, and qPCR when measuring differential expression of
microRNAs
Pradervand S, Weber J, Lemoine F, Consales F, Paillusson A, Dupasquier
M, Thomas J, Richter H, Kaessmann H, Beaudoing E, Hagenbüchle O,
Harshman K.
Biotechniques. 2010 48(3): 219-222
Genomic Technologies Facility, Center for Integrative Genomics,
University of Lausanne, Genopode Building, Lausanne, Switzerland

Profiling microRNA (miRNA) expression is of widespread interest given
the critical role of miRNAs in many cellular functions. Profiling can
be achieved via hybridization-based (microarrays), sequencing-based, or
amplification-based (quantitative reverse transcription-PCR, qPCR)
technologies. Among these, microarrays face the significant challenge
of accurately distinguishing between mature and immature miRNA forms,
and different vendors have developed different methods to meet this
challenge. Here we measure differential miRNA expression using the
Affymetrix, Agilent, and Illumina microarray platforms, as well as qPCR
(Applied Biosystems) and ultra high-throughput sequencing (Illumina).
We show that the differential expression measurements are more
divergent when the three types of microarrays are compared than when
the Agilent microarray, qPCR, and sequencing technology measurements
are compared, which exhibit a good overall concordance.Amplification-free digital gene expression
profiling from minute cell quantities
Ozsolak F, Ting DT, Wittner BS, Brannigan BW, Paul S, Bardeesy N,
Ramaswamy S, Milos PM, Haber DA.
Nat Methods. 2010 7(8):619-21. Epub 2010 Jul 18.
Helicos BioSciences Corporation, Cambridge, Massachusetts, USA

Accurate profiling of minute quantities of RNA in a global manner can
enable key advances in many scientific and clinical disciplines. Here,
we present low-quantity RNA sequencing (LQ-RNAseq), a high-throughput
sequencing-based technique allowing whole transcriptome surveys from
subnanogram RNA quantities in an amplification/ligation-free manner.
LQ-RNAseq involves first-strand cDNA synthesis from RNA templates,
followed by 3' polyA tailing of the single-stranded cDNA products and
direct single molecule sequencing. We applied LQ-RNAseq to profile S.
cerevisiae polyA+ transcripts, demonstrate the reproducibility of the
approach across different sample preparations and independent
instrument runs, and establish the absolute quantitative power of this
method through comparisons with other reported transcript profiling
techniques and through utilization of RNA spike-in experiments. We
demonstrate the practical application of this approach to define the
transcriptional landscape of mouse embryonic and induced pluripotent
stem cells, observing transcriptional differences, including over 100
genes exhibiting differential expression between these otherwise very
similar stem cell populations. This amplification-independent
technology, which utilizes small quantities of nucleic acid and
provides quantitative measurements of cellular transcripts, enables
global gene expression measurements from minute amounts of materials
and offers broad utility in both basic research and translational
biology for characterization of rare cells.Single-molecule sequencing of an individual
human genome
Pushkarev D, Neff NF, Quake SR.
Nat Biotechnol. 2009 27(9): 847-852
Department of Bioengineering, Stanford University and Howard Hughes
Medical Institute, Stanford, California, USA.

Recent advances in high-throughput DNA sequencing technologies have
enabled order-of-magnitude improvements in both cost and throughput.
Here we report the use of single-molecule methods to sequence an
individual human genome. We aligned billions of 24- to 70-bp reads (32
bp average) to approximately 90% of the National Center for
Biotechnology Information (NCBI) reference genome, with 28x average
coverage. Our results were obtained on one sequencing instrument by a
single operator with four data collection runs. Single-molecule
sequencing enabled analysis of human genomic information without the
need for cloning, amplification or ligation. We determined
approximately 2.8 million single nucleotide polymorphisms (SNPs) with a
false-positive rate of less than 1% as validated by Sanger sequencing
and 99.8% concordance with SNP genotyping arrays. We identified 752
regions of copy number variation by analyzing coverage depth alone and
validated 27 of these using digital PCR. This milestone should allow
widespread application of genome sequencing to many aspects of genetics
and human health, including personal genomics.Digital
PCR on a SlipChip
Shen F, Du W, Kreutz JE, Fok A, Ismagilov RF.
Lab Chip. 2010 10(20):2666-72. Epub 2010 Jul 1.
Department of Chemistry and Institute for Biophysical Dynamics, The
University of Chicago, 929 E 57th St, Chicago, Illinois 60637, USA

This paper describes a SlipChip to perform digital PCR in a very simple
and inexpensive format. The fluidic path for introducing the sample
combined with the PCR mixture was formed using elongated wells in the
two plates of the SlipChip designed to overlap during sample loading.
This fluidic path was broken up by simple slipping of the two plates
that removed the overlap among wells and brought each well in contact
with a reservoir preloaded with oil to generate 1280 reaction
compartments (2.6 nL each) simultaneously. After thermal cycling,
end-point fluorescence intensity was used to detect the presence of
nucleic acid. Digital PCR on the SlipChip was tested quantitatively by
using Staphylococcus aureus genomic DNA. As the concentration of the
template DNA in the reaction mixture was diluted, the fraction of
positive wells decreased as expected from the statistical analysis. No
cross-contamination was observed during the experiments. At the
extremes of the dynamic range of digital PCR the standard confidence
interval determined using a normal approximation of the binomial
distribution is not satisfactory. Therefore, statistical analysis based
on the score method was used to establish these confidence intervals.
The SlipChip provides a simple strategy to count nucleic acids by using
PCR. It may find applications in research applications such as single
cell analysis, prenatal diagnostics, and point-of-care diagnostics.
SlipChip would become valuable for diagnostics, including applications
in resource-limited areas after integration with isothermal nucleic
acid amplification technologies and visual readout.Somatic deletion of the NF1 gene in a
neurofibromatosis type 1-associated malignant melanoma demonstrated by
digital PCR
Rübben A, Bausch B, Nikkels A.
Mol Cancer. 2006 Sep 10;5:36.
Department of Dermatology, University Hospital RWTH Aachen,
Pauwelsstrasse 30, D-52074 Aachen, Germany

BACKGROUND: Neurofibromatosis
type 1 (NF1) is the most common hereditary neurocutaneous disorder and
it is associated with an elevated risk for malignant tumors of tissues
derived from neural crest cells. The NF1 gene is considered a tumor
suppressor gene and inactivation of both copies can be found in
NF1-associated benign and malignant tumors. Melanocytes also derive
from neural crest cells but melanoma incidence is not markedly elevated
in NF1. In this study we could analyze a typical superficial spreading
melanoma of a 15-year-old boy with NF1 for loss of heterozygosity (LOH)
within the NF1 gene. Neurofibromatosis in this patient was transmitted
by the boy's farther who carried the mutation NF1 c. 5546 G/A.RESULTS: Melanoma cells
were isolated from formalin-fixed tissue by liquid coverslip laser
microdissection. In order to obtain statistically significant LOH data,
digital PCR was performed at the intragenic microsatellite IVS27AC28
with DNA of approx. 3500 melanoma cells. Digital PCR detected 23
paternal alleles and one maternal allele. Statistical analysis by SPRT
confirmed significance of the maternal allele loss.CONCLUSION: To our
knowledge, this is the first molecular evidence of inactivation of both
copies of the NF1 gene in a typical superficial spreading melanoma of a
patient with NF1. The classical double-hit inactivation of the NF1 gene
suggests that the NF1 genetic background promoted melanoma genesis in
this patient.Taking
qPCR to a higher level: Analysis of CNV reveals the power of
high throughput qPCR to enhance quantitative resolution
Suzanne Weaver, Simant Dube, Alain Mir, Jian Qin,
Gang Sun, Ramesh Ramakrishnan, Robert C. Jones, Kenneth J. Livak
Methods. 2010 Apr;50(4):271-6. Epub 2010 Jan 15.
Fluidigm Corporation, 7000 Shoreline Court, Suite 100, South San
Francisco, CA 94080, USA.

This paper assesses the quantitative resolution of qPCR using copy
number variation (CNV) as a paradigm. An error model is developed for
real-time qPCR data showing how the precision of CNV determination
varies with the number of replicates. Using samples with varying
numbers of X chromosomes, experimental data demonstrates that real-time
qPCR can readily distinguish four copes from five copies, which
corresponds to a 1.25-fold difference in relative quantity. Digital PCR
is considered as an alternative form of qPCR. For digital PCR, an error
model is shown that relates the precision of CNV determination to the
number of reaction chambers. The quantitative capability of digital PCR
is illustrated with an experiment distinguishing four and five copies
of the human gene MRGPRX1. For either real-time qPCR or digital PCR,
practical application of these models to achieve enhanced quantitative
resolution requires use of a high throughput PCR platform that can
simultaneously perform thousands of reactions. Comparing the two
methods, real-time qPCR has the advantage of throughput and digital PCR
has the advantage of simplicity in terms of the assumptions made for
data analysis.One bacterial cell, one complete genome
Woyke T, Tighe D, Mavromatis K, Clum A, Copeland A, Schackwitz W,
Lapidus A, Wu D, McCutcheon JP, McDonald BR, Moran NA, Bristow J, Cheng
JF.
PLoS One. 2010 5(4): e10314
Department of Energy Joint Genome Institute, Walnut Creek, California,
USA

While the bulk of the finished microbial genomes sequenced to date are
derived from cultured bacterial and archaeal representatives, the vast
majority of microorganisms elude current culturing attempts, severely
limiting the ability to recover complete or even partial genomes from
these environmental species. Single cell genomics is a novel
culture-independent approach, which enables access to the genetic
material of an individual cell. No single cell genome has to our
knowledge been closed and finished to date. Here we report the
completed genome from an uncultured single cell of Candidatus Sulcia
muelleri DMIN. Digital PCR on single symbiont cells isolated from the
bacteriome of the green sharpshooter Draeculacephala minerva bacteriome
allowed us to assess that this bacteria is polyploid with genome copies
ranging from approximately 200-900 per cell, making it a most suitable
target for single cell finishing efforts. For single cell shotgun
sequencing, an individual Sulcia cell was isolated and whole genome
amplified by multiple displacement amplification (MDA). Sanger-based
finishing methods allowed us to close the genome. To verify the
correctness of our single cell genome and exclude MDA-derived
artifacts, we independently shotgun sequenced and assembled the Sulcia
genome from pooled bacteriomes using a metagenomic approach, yielding a
nearly identical genome. Four variations we detected appear to be
genuine biological differences between the two samples. Comparison of
the single cell genome with bacteriome metagenomic sequence data
detected two single nucleotide polymorphisms (SNPs), indicating
extremely low genetic diversity within a Sulcia population. This study
demonstrates the power of single cell genomics to generate a complete,
high quality, non-composite reference genome within an environmental
sample, which can be used for population genetic analyzes.Spinning disk platform for microfluidic
digital polymerase chain reaction.
Sundberg SO, Wittwer CT, Gao C, Gale BK.
Anal Chem. 2010 82(4): 1546-1550.
University of Utah, Rm 5R441, 1795 E South Campus Dr., Salt Lake City,
Utah 84112, USA

An inexpensive plastic disk disposable was designed for digital
polymerase chain reaction (PCR) applications with a microfluidic
architecture that passively compartmentalizes a sample into 1000
nanoliter-sized wells by centrifugation. Well volumes of 33 nL were
attained with a 16% volume coefficient of variation (CV). A rapid air
thermocycler with aggregate real-time fluorescence detection was used,
achieving PCR cycle times of 33 s and 94% PCR efficiency, with a
melting curve to validate product specificity. A CCD camera acquired a
fluorescent image of the disk following PCR, and the well intensity
frequency distribution and Poisson distribution statistics were used to
count the positive wells on the disk to determine the number of
template molecules amplified. A 300 bp plasmid DNA product was
amplified within the disk and analyzed in 50 min with 58-1000 wells
containing plasmid template. Target concentrations measured by the
spinning disk platform were 3 times less than that predicted by
absorbance measurements. The spinning disk platform reduces disposable
cost, instrument complexity, and thermocycling time compared to other
current digital PCR platforms.